HIV type 1 (HIV-1) and HIV type 2 (HIV-2) genomes differ by about 50–60% at the nucleotide level. Such differences may be correlated with a differential response to some antiretrovirals, as observed with the natural resistance of HIV-2 to non-nucleoside reverse transcriptase inhibitors (NNRTIs) and to the fusion inhibitor enfuvirtide, and the decreased susceptibility to some of protease inhibitors [1–3]. Previous studies suggest comparable HIV-1 and HIV-2 in-vitro susceptibility to integrase inhibitors (INIs) such as raltegravir and elvitegravir, with a potent in-vivo clinical and virological response for raltegravir in HIV-2-infected patients [4–6]. A new drug in this class, S/GSK1349572, a once-daily unboosted INI showing potent antiviral activity in HIV-1-infected patients, is currently in development . The impact of HIV genetic diversity within the integrase gene on the antiviral activity of S/GSK1349572 is currently unknown.
Herein, we assessed the in-vitro susceptibility of HIV-2 wild type and HIV-2 integrase-mutated clinical isolates to the new INI compound S/GSK1349572.
Phenotypic susceptibility to S/GSK1349572 was determined using the Agence Nationale de Recherches sur le SIDA et les hépatites virales (ANRS) peripheral blood mononuclear cells (PBMCs) method, as previously described . Phenotypic assay was performed on: ROD HIV-2 reference strain; BRU HIV-1 reference strain; co-cultivated HIV-2 isolates obtained from nine INI-naïve HIV-2-infected patients; and co-cultivated HIV-2 isolates harboring raltegravir resistance-associated mutations obtained from two HIV-2-infected raltegravir-experienced patients. One of these latter patients had provided two sequential samples. Briefly, after HIV-2 isolation from PBMC, the cell-free HIV-2-positive supernatant was serially diluted (100–10−2) and incubated with fresh normal phytohemagglutinin-stimulated PBMC. After being washed, the cells were placed in 96-well plates containing six serial dilutions of the antiretroviral drug. Each dilution was tested in quadruplicate. On day 3, the supernatant was collected and the 50% tissue culture-infective dose (TCID50) was assessed by measuring the number of HIV-2 RNA copies in the supernatant with a real-time quantitative reverse transcription-PCR assay . Drug concentrations inhibiting the replication of 100 TCID50 by 50% and 90% were calculated (EC50 and EC90). Integrase gene direct sequencing was performed in all INI-naïve patient specimens both on plasma samples and on supernatant at time of phenotypic assay.
HIV-2 subtypes isolated from patients were subtype A (n = 8), B (n = 2) and H (n = 1). No raltegravir resistance-associated mutations, primary or secondary among the following H51Y; T66I/A/K; V72I; L74I/A/M; E92Q; T97A; T112I; F121Y; T125K; A128T; E138K/A/D; G140A/S/R/C/H; Y143C/H/R; Q146K/P; S147G; Q148K/R/H; V151I; S153Y/A; M154I; N155S/H; K156N; E157Q; K160D/N; G163R/K; V165I; V201I; I203M; T206S; S230N/R; V249I; R263K; and C280Y  were detected by direct sequencing in samples issued from INI-naïve patients. One of the INI-experienced patients had plasma virus with T97A and Y143C raltegravir resistance-associated mutations. The remaining INI-experienced patient had plasma virus with G140S and Q148R raltegravir resistance-associated mutations at the first assessment. As described in HIV-1 , an evolution of genotypic resistance profile was observed at the second time point assessed in this patient, with a switch from G140S to G140T and the selection of N155H. These mutations might be harbored on different viral genomes, as previously shown for HIV-1 . Of note, the G140T substitution is an uncommon residue at this position in HIV-1 with unknown phenotypic effect in that genetic context.
No changes were observed in integrase gene for all of the HIV-2 clinical isolates between day 0 and day 3 of the ANRS PBMC phenotypic susceptibility assay. Integrase sequences obtained from INI-naïve HIV-2-infected patients showed integrase gene polymorphisms involving similar positions as already described in our previous study  (data not shown).
Median EC50 and EC90 values to S/GSK1349572 for the nine tested HIV-2 INI-naïve isolates were 0.8 nM (range 0.2–1.4) and 7 nM (range 6–16), respectively. These values were similar to those observed for HIV-2 ROD reference strain showing EC50 and EC90 values of 2 and 21 nM, respectively, as well as the HIV-1 BRU reference strain, with EC50 and EC90 values of 4 and 13 nM . When assessing integrase-mutated variants, we found EC50 values to S/GSK1349572 were increased by 13- and 18-fold for the HIV-2 subtype A samples with double (G140S + Q148R) and triple (G140T + Q148R + N155H) mutations, respectively, compared to INI-naïve samples EC50 values. The subtype H double mutant isolate exhibiting the T97A + Y143C INI resistance-associated mutations showed a seven-fold increase in EC50 values compared to INI-naïve strains EC50 values. EC50 values to raltegravir of the G140S + Q148R, G140T + Q148R + N155H, and T97A + Y143C integrase mutant samples were 165 nM (fold change = 63), 24 nM (fold change = 9), and 11 nM (fold change = 5), respectively.
This is the first study to assess the in-vitro susceptibility of HIV-2 clinical isolates, including integrase-mutant isolates, to the new INI compound S/GSK1349572. EC50 values obtained for the INI-naïve HIV-2 isolates were in a similar range to those previously shown for HIV-2 to raltegravir and elvitegravir  and were consistent with those previously published for HIV-1 . Thus, similar to raltegravir or elvitegravir, this new compound of the INI drug class should represent a new therapeutic option for HIV-2-infected patients. The activity of S/GSK1349572 is now being assessed in phase 2b multicenter clinical studies.
The increased EC50 values to S/GSK1349572 observed in the double or triple integrase-mutated viruses with the Q148R mutation tested in this series were consistent with the effects of these mutations in the context of HIV-1 integrase . The HIV-1 double mutant T97A + Y143C was not tested in that latter study . Unfortunately, no HIV-2 clinical isolate harboring a single raltegravir resistance-associated mutation was available to be tested in our study because such viruses failed to be co-cultured with PBMC. Thus, further investigations on clinical isolates or site-directed mutants harboring only one INI resistance-associated mutation are needed to better characterize the resistance profile of the S/GSK1349572 in the context of HIV-2 integrase, and to clinically validate the expectation for the limited cross-resistance to raltegravir and elvitegravir and the improved resistance profile of this compound  in HIV-2-infected patients.
Taken together, our findings showed that S/GSK1349572 is active against wild-type HIV-2 viruses. More data are needed to reliably assess S/GSK1349572 activity against HIV-2 INI-resistant viruses.
The research leading to these results has received funding from the French National Agency for Research on AIDS and Viral Hepatitis (ANRS) and the European Community's Seventh Framework Program (FP7/2007–2013) under the project ‘Collaborative HIV and Anti-HIV Drug Resistance Network (CHAIN)’ (grant no. 223131). We also thank Shionogi-ViiV Healthcare LLC for the compound S/GSK1349572 and financial support.
Authors' contribution: C.C., F.D., F.B.V. and D.D. contributed to the study concept. G.Co. performed the phenotypic and genotypic tests. C.C., L.L., F.D., S.M., G.Ch., T.N., R.S. and D.D. contributed to the analysis and interpretation of the data. C.C., F.D., R.S., and D.D. contributed to the writing of the manuscript. All authors contributed to the critical review of the manuscript.
1. Desbois D, Roquebert B, Peytavin G, Damond F, Collin G, Bénard A, et al
. In vitro phenotypic susceptibility of human immunodeficiency virus type 2 clinical isolates to protease inhibitors. Antimicrob Agents Chemother 2008; 52:1545–1548.
2. Witvrouw M, Pannecouque C, Switzer WM, Folks TM, De Clercq E, Heneine W. Susceptibility of HIV-2, SIV and SHIV to various anti-HIV-1 compounds: implications for treatment and postexposure prophylaxis. Antivir Ther 2004; 9:57–65.
3. Tantillo C, Ding J, Jacobo-Molina A, Nanni RG, Boyer PL, Hughes SH, et al
. Locations of anti-AIDS drug binding sites and resistance mutations in the three-dimensional structure of HIV-1 reverse transcriptase. Implications for mechanisms of drug inhibition and resistance. J Mol Biol 1994; 243:369–387.
4. Roquebert B, Damond F, Collin G, Matheron S, Peytavin G, Bénard A, et al
. HIV-2 integrase gene polymorphism and phenotypic susceptibility of HIV-2 clinical isolates to the integrase inhibitors raltegravir and elvitegravir in vitro. J Antimicrob Chemother 2008; 62:914–920.
5. Garrett N, Xu L, Smit E, Ferns B, El-Gadi S, Anderson J. Raltegravir treatment response in an HIV-2 infected patient: a case report. AIDS 2008; 22:1091–1092.
6. Salgado M, Toro C, Simón A, Garrido C, Blanco F, Soriano V, et al
. Mutation N155H in HIV-2 integrase confers high phenotypic resistance to raltegravir and impairs replication capacity. J Clin Virol 2009; 46:173–175.
7. Lalezari J, Sloan L, Dejesus E, Hawkins T, Mccurdy L, Song I, et al. Potent antiviral activity of S/GSK1349572, a next generation integrase inhibitor (INI), in INI-naïve HIV-1-infected patients
[Abstract TUAB105]. In: 5th IAS Conference on HIV Pathogenesis, Treatment and Prevention
. Cape Town, South Africa, July 2009.
8. Damond F, Collin G, Descamps D, Matheron S, Pueyo S, Taieb A, et al
. Improved sensitivity of human immunodeficiency virus type 2 subtype B plasma viral load assay. J Clin Microbiol 2005; 43:4234–4236.
9. Lataillade M, Chiarella J, Kozal MJ. Natural polymorphism of the HIV-1 integrase gene and mutations associated with integrase inhibitor resistance. Antivir Ther 2007; 12:563–570.
10. Malet I, Delelis O, Soulie C, Wirden M, Tchertanov L, Mottaz P, et al
. Quasispecies variant dynamics during emergence of resistance to raltegravir in HIV-1-infected patients. J Antimicrob Chemother 2009; 63:795–804.
11. Quercia R, Dam E, Perez-Bercoff D, Clavel F. Selective-advantage profile of human immunodeficiency virus type 1 integrase mutants explains in vivo evolution of raltegravir resistance genotypes. J Virol 2009; 83:10245–10249.
12. Sato A, Kobayashi M, Yoshinaga T, Fujiwara T, Underwood M, Johns B, et al. S/GSK1349572 is a potent next generation HIV integrase inhibitor
[Abstract WEPEA097]. In: 5th IAS Conference on HIV Pathogenesis, Treatment and Prevention
. Cape Town, South Africa, July 2009.
13. Seki T, Kobayashi M, Wakasa-Morimoto C, Yoshinaga T, Sato A, Fujiwara T, et al. S/GSK1349572 is a potent next generation HIV integrase inhibitor and demonstrates a superior resistance profile substantiated with 60 integrase mutant molecular clones
[Abstract 555]. In: 17th Conference on Retroviruses and Opportunistic Infections
. San Francisco, CA, USA, February 2010.
14. Underwood M, Johns B, Sato A, Fujiwara T, Spreen W. S/GSK1349572: a next generation integrase inhibitor with activity against integrase inhibitor resistant clinical isolates from patients experiencing virologic failure while on raltegravir therapy
[Abstract WEPEA098]. In: 5th IAS Conference on HIV Pathogenesis, Treatment and Prevention
. Cape Town, South Africa, July 2009.